Subunit vaccine for swine fever and preparation method thereof

Abstract

The invention discloses a hog cholera subunit vaccine and a preparation method thereof, and belongs to the technical field of hog cholera subunit vaccines.The hog cholera subunit vaccine is a pure-line hog cholera lapinized virus strain CSFV CC-1 which is highly adaptive to rabbit source cells or common cells, single in genotype and stable in inheritance and is obtained through a hog cholera lapinized virus strain through a virus dilution technology. The swine fever subunit vaccine is developed by matching the treated and extracted virus subunit antigen stock solution with the subunit antigen adjuvant, so that the possibilities of virus dispersion, homologous recombination and variation can be effectively reduced; the immunogenicity of antigens is enhanced, the onset time of immunoreaction is shortened, pigs can be effectively prevented and protected from being infected by hog cholera viruses, the infection rate and breeding loss are greatly reduced, infected animals and immune animals can be effectively distinguished, and the elimination of potential hog cholera viruses in pig farms is effectively accelerated.

Description

Technical Field

[0001] The present invention relates to the technical field of swine fever subunit vaccines, and more specifically, to a swine fever subunit vaccine and a preparation method thereof. Background Art

[0002] Classical swine fever, commonly known as "rotten intestinal plague", is an acute, febrile, contact infectious disease caused by the swine fever virus of the genus Classical swine fever virus of the family Flaviviridae. It is a type of infectious disease and parasitic disease. It is highly contagious and lethal.

[0003] At present, in the process of treatment and protection against classical swine fever virus, classical swine fever subunit vaccines are needed for prevention and treatment. However, the existing vaccines have weak cellular immunity and certain side effects. They cannot produce effective antibodies and protection for pigs against viral infection. In addition, the existing vaccines are prone to toxin shedding, homologous recombination and mutation, which is not conducive to enhancing the immunogenicity of antigens and shortening the time for the immune response to take effect. Therefore, it is necessary to study a classical swine fever subunit vaccine and its preparation method. Summary of the invention

[0004] (1) Technical issues to be solved

[0005] In view of the shortcomings of the prior art, the object of the present invention is to provide a classical swine fever subunit vaccine and a preparation method thereof, which has the characteristics of being able to produce effective antibodies and having good preventive properties.

[0006] (2) Technical solution

[0007] To achieve the above object, the present invention provides a swine fever subunit vaccine, comprising:

[0008] The classical swine fever subunit vaccine uses the classical swine fever rabbit-attenuated strain CSFV CC-1, which is highly adapted to rabbit-derived cells or common cells, has a single genotype and can be stably inherited, obtained through virus dilution technology. After cell culture and harvesting of the virus liquid, stabilizers and adjuvants are added after dilution to produce the classical swine fever subunit vaccine.

[0009] A method for preparing a swine fever subunit vaccine comprises the following preparation steps:

[0010] S1. Cell subculture and culture for production

[0011] After the seed cells are digested and dispersed, they are cultured and used for continued passage or virus inoculation when they form a monolayer or grow to a suitable density;

[0012] S2. Reproduction of seeds

[0013] CSFV CC-1 seed batch virus is inoculated with rabbit-derived cells or seed batch cells of commonly used cells with good production, and the virus is collected after cultivation to be used as the seed virus for production;

[0014] S3. Propagation of virus fluid for production

[0015] The production virus is inoculated into the well-growing production cells for cell culture. After the cells are fully grown, the cell maintenance medium is replaced and the culture is continued. The virus liquid is harvested and replaced, and the virus liquid is harvested and stored as the virus liquid for seedling preparation to obtain the virus antigen. A lysing agent is added, and a protein hydrolysis method is used to decompose the complex structure of the virus antigen, extract the special protein structure of bacteria and viruses, and screen out the fragments with immune activity to obtain a specific subunit antigen stock solution;

[0016] S4. Selection and preparation of subunit vaccine adjuvants

[0017] 50% by mass of plant essential oil, 12% of soybean oil lecithin and 5.5% of saponin are magnetically suspended and stirred for 60 minutes, then 12.5% ​​of Span 80, 5% of Tween 80 and 12% of propylene glycol are added to the uniformly mixed plant oil at once, magnetically suspended and stirred for 20 minutes, then 3% of astragalus polysaccharide aqueous solution is added, stirred until clear and transparent and then allowed to stand to obtain a subunit antigen adjuvant;

[0018] S5. Seedling preparation and packaging

[0019] The virus subunit antigen stock solution that has passed the inspection is mixed with the subunit adjuvant antigen in proportion, and a stabilizer and an antibiotic are added, mixed evenly, and then quantitatively packaged to obtain the swine fever subunit vaccine.

[0020] A classical swine fever subunit vaccine according to the present technical scheme is developed by processing the extracted virus subunit antigen stock solution and combining it with a subunit antigen adjuvant. It can effectively reduce the possibility of virus shedding, homologous recombination and mutation, enhance the immunogenicity of the antigen, shorten the time for the immune response to take effect, prevent and protect the pig herd from infection with classical swine fever virus, greatly reduce the infection rate and breeding losses, and ensure the stability of the vaccine through multiple groups of vaccine samples and multiple groups of experiments after piglet injection and virus vaccination, detect virus antibodies in the piglets after vaccination, improve the survival rate of piglets, and reduce production costs.

[0021] Furthermore, the culture time in S1 and the culture time in S3 are both 3-4 days, and the frequency of harvesting and changing the medium in S3 is once every 2-4 days, for a total of 10-15 times.

[0022] Furthermore, the storage temperature of the virus solution in S3 is -15°C.

[0023] Furthermore, commonly used cells in S2 include but are not limited to bovine testicular primary cells, sheep testicular primary cells, sheep kidney primary cells, bovine BT cell line, porcine SK-6 cell line, etc.

[0024] Furthermore, the cell culture in S1 refers to the culture of cells at a suitable temperature and density by roller bottle culture, cell microcarrier culture or cell suspension culture.

[0025] Furthermore, the cells in S1 are dispersed using a trypsin dispersion medium, and the cell maintenance medium in S3 is a low-serum culture medium containing newborn calf serum.

[0026] Furthermore, during the process of harvesting the venom solution for seedling preparation, production cells are added when the solution is harvested and replaced.

[0027] (3) Beneficial effects

[0028] In summary, the present invention has the following beneficial effects:

[0029] The present invention has the advantages of high batch-to-batch stability of product quality, and at the same time has the advantages of strong immunogenicity and good safety. The swine fever subunit vaccine is developed by processing the extracted virus subunit antigen stock solution and cooperating with the subunit antigen adjuvant, which can effectively reduce the possibility of virus shedding, homologous recombination and mutation, enhance the immunogenicity of the antigen, shorten the time for the immune response to take effect, and can effectively prevent and protect the pig herd from infection by swine fever virus, greatly reducing the infection rate and breeding losses. Through multiple groups of vaccine samples and multiple groups of experiments after piglets are injected with the virus, the stability of the vaccine is guaranteed and virus antibodies are detected in the piglets after the injection of the vaccine, the survival rate of the piglets is improved, and the production cost is reduced. Compared with general inactivated vaccines, the present application can effectively distinguish between infected animals and immune animals, and effectively accelerate the removal of potential swine fever viruses in pig farms. DETAILED DESCRIPTION

[0030] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] The present invention provides a technical solution: a swine fever subunit vaccine, comprising:

[0032] The classical swine fever subunit vaccine uses the classical swine fever rabbit-attenuated strain CSFV CC-1, which is highly adapted to rabbit-derived cells or common cells, has a single genotype and can be stably inherited, obtained through virus dilution technology. After cell culture and harvesting of the virus liquid, stabilizers and adjuvants are added after dilution to produce the classical swine fever subunit vaccine.

[0033] A method for preparing a swine fever subunit vaccine, characterized in that it comprises the following preparation steps:

[0034] S1. Cell subculture and culture for production

[0035] After the seed cells are digested and dispersed, they are cultured and used for continued passage or virus inoculation when they form a monolayer or grow to a suitable density;

[0036] S2. Reproduction of seeds

[0037] CSFV CC-1 seed batch virus is inoculated with rabbit-derived cells or seed batch cells of commonly used cells with good production, and the virus is collected after cultivation to be used as the seed virus for production;

[0038] S3. Propagation of virus fluid for production

[0039] The production virus is inoculated into the well-growing production cells for cell culture. After the cells are fully grown, the cell maintenance medium is replaced and the culture is continued. The virus liquid is harvested and replaced, and the virus liquid is harvested and stored as the virus liquid for seedling preparation to obtain the virus antigen. A lysing agent is added, and a protein hydrolysis method is used to decompose the complex structure of the virus antigen, extract the special protein structure of bacteria and viruses, and screen out the fragments with immune activity to obtain a specific subunit antigen stock solution;

[0040] S4, taking 50% by mass of plant essential oil, 12% of soybean oil lecithin and 5.5% of saponin, magnetically stirring for 60 minutes, then taking 12.5% ​​of Span 80, 5% of Tween 80 and 12% of propylene glycol, adding them to the mixed vegetable oil at once, magnetically stirring for 20 minutes, then adding 3% of astragalus polysaccharide aqueous solution, stirring until clear and transparent, and then standing to obtain a subunit antigen adjuvant;

[0041] S5. Seedling preparation and packaging

[0042] The virus subunit antigen stock solution that has passed the inspection is mixed with the subunit adjuvant antigen in proportion, and a stabilizer and an antibiotic are added, mixed evenly, and then quantitatively packaged to obtain the swine fever subunit vaccine.

[0043] Specifically, the culture time in S1 and the culture time in S3 are both 3-4 days, and the frequency of harvesting and changing the medium in S3 is once every 2-4 days, for a total of 10-15 times.

[0044] Specifically, the storage temperature of the virus solution in S3 is -15°C.

[0045] Specifically, commonly used cells in S2 include, but are not limited to, bovine testicular primary cells, sheep testicular primary cells, sheep kidney primary cells, bovine BT cell line, porcine SK-6 cell line, and the like.

[0046] Specifically, the cell culture in S1 refers to the culture of cells at a suitable temperature and density through roller bottle culture, cell microcarrier culture or cell suspension culture.

[0047] Specifically, the cells in S1 are dispersed using a trypsin dispersion solution, and the cell maintenance solution in S3 is a low-serum culture medium containing newborn calf serum. During the process of harvesting the venom solution for seedling preparation, the production cells are supplemented when the solution is harvested and replaced.

[0048] By adding stabilizers and antibiotics, the temperature resistance of the vaccine can be increased, which facilitates storage and transportation. At the same time, the effectiveness of the vaccine can be guaranteed, the preventive effect can be increased, which is beneficial for the prevention of swine fever virus in pig herds.

[0049] Example

[0050] 1. Vaccine sample preparation

[0051] The virus liquid that passes the test is mixed with a stabilizer and an antibiotic in proportion, and a vaccine adjuvant is added and stirred to obtain a vaccine sample.

[0052] Sample 1:

[0053] The vaccine was prepared by the above method, except that the virus liquid was mixed with the stabilizer and the antibiotic in a molar ratio of 1:1.2:1.3, which was used as sample 1.

[0054] Sample 2:

[0055] The inactivated vaccine was prepared by the above method, except that the virus liquid was mixed with the stabilizer and the antibiotic in a molar ratio of 2:1.3:1.2, which was used as sample 2.

[0056] Sample 3:

[0057] The inactivated vaccine was prepared by the above method, except that the virus liquid was mixed with the stabilizer and the antibiotic in a molar ratio of 2.5:2:3, as sample 3.

[0058] Sample 4:

[0059] The inactivated vaccine was prepared by the above method, except that the virus liquid was mixed with the stabilizer and the antibiotic in a molar ratio of 3:2:2.3, which was used as sample 4.

[0060] 1.1 Vaccine abnormal toxicity detection

[0061] Twenty-five 28-day-old piglets were randomly divided into five groups, with five piglets in each group raised in isolation. Four groups of piglets were injected with vaccines intramuscularly behind the ear roots, and one group was the control group, which was not vaccinated. The changes in body temperature, mental and appetite status, and body bleeding of the piglets were recorded. Blood was collected from each group of piglets for antibody testing 28 days after vaccination.

[0062] Table 1: Changes in body temperature of 28-day-old piglets after vaccination (1-14 days)

[0063]

[0064]

[0065] Table 2: Mental, appetite and body bleeding of piglets injected with vaccines on day 28 (1-14 days)

[0066]

[0067] As shown in Table 1 and Table 2, the observation results after the vaccination of piglets showed that the vaccinated pigs did not show an increase in body temperature, and the average temperature increase did not exceed 1°C. In addition, all piglets had normal appetite and spirit after vaccination, and no other visible clinical reactions. It can be judged that this vaccine meets the requirements of the abnormal toxicity test method of Part III of the Pharmacopoeia of the People's Republic of China.

[0068] 2.2 Immunological study

[0069] Each group of piglets in the above experimental groups 1-4 was injected with strong poison. After the poison challenge, the high fever changes, appetite, and body bleeding symptoms of the piglets were recorded, as shown in Table 2

[0070] Table 3: Results of the 28-day piglet immune challenge experiment

[0071]

[0072] As shown in Table 3, the mortality rate of piglets challenged with virus after vaccination in the experimental group and the control group was higher in the control group that was not vaccinated. The vaccination provided 100% protection to the piglets in the experimental group, which greatly reduced the morbidity and mortality of piglets caused by virus infection and enhanced the preventive effect.

[0073] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A swine fever subunit vaccine, characterized in that: include: The classical swine fever subunit vaccine uses the classical swine fever rabbit-attenuated strain CSFV CC-1, which is highly adapted to rabbit-derived cells or common cells, has a single genotype and can be stably inherited, obtained through virus dilution technology. After cell culture and harvesting of the virus liquid, stabilizers and adjuvants are added after dilution to produce the classical swine fever subunit vaccine.

2. The method for preparing a swine fever subunit vaccine according to claim 1, characterized in that: The method comprises the following preparation steps: S1. Cell subculture and culture for production After the seed cells are digested and dispersed, they are cultured and used for continued passage or virus inoculation when they form a monolayer or grow to a suitable density; S2. Reproduction of seeds CSFV CC-1 seed batch virus is inoculated with rabbit-derived cells or seed batch cells of commonly used cells with good production, and the virus is collected after cultivation to be used as the seed virus for production; S3. Propagation of virus fluid for production The production virus is inoculated into the well-growing production cells for cell culture. After the cells are fully grown, the cell maintenance medium is replaced and the culture is continued. The virus liquid is harvested and replaced, and the virus liquid is harvested and stored as the virus liquid for seedling preparation to obtain the virus antigen. A lysing agent is added, and a protein hydrolysis method is used to decompose the complex structure of the virus antigen, extract the special protein structure of bacteria and viruses, and screen out the fragments with immune activity to obtain a specific subunit antigen stock solution; S4. Selection and preparation of subunit vaccine adjuvants 50% by mass of plant essential oil, 12% of soybean oil lecithin and 5.5% of saponin are magnetically suspended and stirred for 60 minutes, then 12.5% ​​of Span 80, 5% of Tween 80 and 12% of propylene glycol are added to the evenly mixed vegetable oil in sequence, magnetically suspended and stirred for 20 minutes, and then 3% of astragalus polysaccharide aqueous solution is added, stirred until clear and transparent and then allowed to stand to obtain a subunit antigen adjuvant. S5. Seedling preparation and packaging The virus subunit antigen stock solution that has passed the inspection is mixed with the subunit adjuvant antigen in proportion, and a stabilizer and an antibiotic are added, mixed evenly, and then quantitatively packaged to obtain the swine fever subunit vaccine.

3. The method for preparing a swine fever subunit vaccine according to claim 2, characterized in that: The culture time in S1 and S3 is 3-4 days. The frequency of harvesting and changing the medium in S3 is once every 2-4 days, for a total of 10-15 times.

4. The method for preparing a swine fever subunit vaccine according to claim 1, characterized in that: The virus solution in S3 was stored at -15°C.

5. The method for preparing a swine fever subunit vaccine according to claim 2, characterized in that: Commonly used cells in S2 include, but are not limited to, bovine testicular primary cells, sheep testicular primary cells, sheep kidney primary cells, bovine BT cell line, porcine SK-6 cell line, etc.

6. The method for preparing a swine fever subunit vaccine according to claim 2, characterized in that: The cell culture in S1 refers to the culture of cells at a suitable temperature and density by roller bottle culture, cell microcarrier culture or cell suspension culture.

7. The method for preparing a swine fever subunit vaccine according to claim 1, characterized in that: The cells in S1 were dispersed using trypsin dispersion medium, and the cells in S3 were maintained using low-serum culture medium containing newborn calf serum.

8. The method for preparing a swine fever subunit vaccine according to claim 3, characterized in that: During the process of harvesting the venom solution for seedling preparation, production cells are added when the solution is replaced.

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